Mark Fellmann, Div. Of Molecular Genome Analysis, German Cancer Research Center, Heidelberg, Germany
Treatment protocol
10 nM 17-beta Estradiol solved in ethanol
Growth protocol
cells were cultivated inRPMI Media (1x) (Invitrogen, Grand Island, NY, USA), supplemented with 10 % fetal calf serum (FCS), 50 U/ml penicillin, and 50 µg/ml streptomycin sulfate, 1% non-essential amino acids (Invitrogen, Grand Island, NY, USA) and 100 µg/ml insulin bovine
Extracted molecule
total RNA
Extraction protocol
total RNA was extracted using Rneasy mini-prep kit (Qiagen, Hilden, Germany; 2µg total RNA was amplified one round based on T7 RNA Polymerase (Agilent low RNA Input Amplification Kit)
Label
Cy3
Label protocol
direct fluorescent labelling of 2 µg amplified RNA, anneling of 0.5µg Random Primer and reverse-transcribed into cDNA with Superscript III reverse transcriptase for 1h at 42°C in the presence of 0.4 mM dGTP, 0.4 mM dATP, 0.4 mM dTTP, 0.24 mM dCTP, 0.125 mM Cy3-dCTP. After hydrolysis of RNA in 0.2 M NaOH, Cy3-labeled probes were purified with Microcon YM-30 column.
Mark Fellmann, Div. Of Molecular Genome Analysis, German Cancer Research Center, Heidelberg, Germany
Treatment protocol
ethanol_control
Growth protocol
cells were cultivated inRPMI Media (1x) (Invitrogen, Grand Island, NY, USA), supplemented with 10 % fetal calf serum (FCS), 50 U/ml penicillin, and 50 µg/ml streptomycin sulfate, 1% non-essential amino acids (Invitrogen, Grand Island, NY, USA) and 100 µg/ml insulin bovine
Extracted molecule
total RNA
Extraction protocol
total RNA was extracted using Rneasy mini-prep kit (Qiagen, Hilden, Germany; 2µg total RNA was amplified one round based on T7 RNA Polymerase (Agilent low RNA Input Amplification Kit)
Label
Cy5
Label protocol
direct fluorescent labelling of 2 µg amplified RNA, anneling of 0.5µg Random Primer and reverse-transcribed into cDNA with Superscript III reverse transcriptase for 1h at 42°C in the presence of 0.4 mM dGTP, 0.4 mM dATP, 0.4 mM dTTP, 0.24 mM dCTP, 0.125 mM Cy5-dCTP. After hydrolysis of RNA in 0.2 M NaOH, Cy5-labeled probes were purified with Microcon YM-30 column.
Hybridization protocol
ch1 and ch2 together are solved in 50 µl 1x DIG-Easy hybridization buffer (Roche Diagnostics, Mannheim, Germany), containing 10x Denhardts solution and 2 ng/µl Cot1-DNA (Invitrogen); sample was denaturated at 65°C for 2min, hybridzation of arrays were performed in Corning chambers 14h at 37°C; slides were washed twice in 1xSSC+0.1SDS and once in 0.1xSSC+ 0.1SDS before air pressure drying and scanning.
Scan protocol
arrays were scanned with the GenePix 4000B microarray scanner and analyzed using GenePix Pro 4.1 software (Axon Instruments)
Description
siRNA_Estradiol_DKFZ_Replicate2
Data processing
raw data processing was performed using ArrayMagic (Buness et al., 2005) including VSN normalization method; after removal of bad quality clones, generalized log ratios from 26618 cDNA clones were given in the data table
